The development of cardiac, vascular and hematopoietic is mutually linked. The hematopoietic cells are not just passengers passively transported through the circulatory system, but substantially involved in the development and repair of the organs. Through the precedent R21 proposal, we have established that endocardial cells give rise to hematopoietic cells. These hemogenic endocardial cells are enriched in the cushion endocardium in mouse embryos and undergo endocardial-hematopoietic transition via Nkx2-5- dependent manner. This mechanism is conserved among species. However, biological significance of this discovery has been hampered by the fact that the endocardially-derived hematopoietic cells rarely contribute to the postnatal hematopoietic stem cell system in the bone marrow. A fundamental question is why the heart needs to generate hematopoietic cells in this specific region at this specific stage. Answer to this questio requires thorough understanding of the molecular mechanism and cellular differentiation of the hemogenic endocardium. This proposal take advantage of specific genetic labeling tool to establish a pivotal Nkx2-5- Notch-Runx1 pathway, a novel tissue macrophage population originating from hemogenic endocardial cells, and a previously unappreciated role of endocardially-derived tissue macrophages in the formation of the cardiac valves. Together, this proposal will provide a biological significance to hemogenic endocardium, and demonstrate that the hemogenic endocardium is not merely a phylogenetical remnant like intestinal cecum but an indispensable component that plays a significant role in the organ function. Congenital valvular anomaly affects 1-2% of live births in the U.S., and patients with genetic predisposition are more likely to develop age- related valvular defects. If funded, this proposal will extend our discovery of hemogenic endocardium to a novel disease mechanism of congenital valvular anomalies.